Maternal-fetal crosstalk. Elucitaing the role of mir30d in endometrial receptivity and pregnancy outcome

Resumen

Reciprocal communication between the endometrium and the preimplantation embryo is required for pregnancy. Our previous work described a novel cell-to-cell communication mechanism involving the delivery of endometrial microRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. More specifically, in vitro transfer of hsa-miR-30d supported mouse embryo adhesion by increasing expression of the adhesion molecules Cdh5, Itβ3, Itα5 in trophectoderm cells. The present doctoral thesis uses a miR-30d KO model to give answer two well-defined objectives: determine the mechanism by which miR-30d is incorporated into the maternal-derived extracellular vesicles and to investigate the impact of maternal or embryonic-origin miR-30d deficiency on implantation and subsequent fetal development. Regarding the first objective, mass spectrometry carried out after performing a co-immunoprecipitation with a biotinylated miR-30d in lysates from human endometrial epithelial cells (hEECs) and their derived exosomes allowed us to identify several proteins that could exert this role within cells. Among them, we found hnRNPC1, a ribonucleoprotein traditionally involved in directing the transfer of mRNAs from the nucleus to the cytoplasm. Co-localization studies of hnRNPC1 with the exosome marker CD63 and FACS analyses demonstrated the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 in an Ishikawa endometrial adenocarcinoma cell line resulted in a sharp decrease of the levels of miR-30d in both epithelial-like cells and exosomes, suggesting its pivotal role in miR-30d biogenesis and transfer. Co-culture assays of miR-30d knock out (KO) embryos with sihnRNPC1 hEECs revealed a decrease in embryo-miR-30d acquisition in the adhesion and invasion stages. The lower adhesion levels observed are evidence that hnRNPC1 is an important player in the maternal-fetal communication established in the early stages of implantation. On the other hand, the endometrial receptivity markers COX2, LIF, MSX1, MSX2, ESR, and PGR were used to examine the impact of miR-30d deficiency, being LIF significantly downregulated in the endometrium of KO mothers. Next, different maternal-embryonic crosstalk scenarios were tested by controlling the origin of miR-30d or its deficiency by transferring WT, miR-30d KO, or KO embryos pre-treated with miR-30d into WT or KO recipients. When transferred into WT recipients, KO embryos had poorer implantation rates (IR) than WT embryos (p = 0.0061). Even lower implantation rates were seen when comparing KO and WT embryos transferred into KO dams (p = 0.0059). Interestingly, a positive correlation (r = 0.9978) was observed for maternal LIF expression and the IR, suggesting dysregulation of LIF associated with the miR-30d knockdown. Also, the course of gestation appeared compromised in KO females as evidenced by smaller implantation sites, higher rates of resorption, and fetuses with smaller crown rump-lengths and FW:PW ratios.